Project Summary Sorting of soluble proteins for transport to intracellular compartments and secretion from cells is essential for cell and tissue homeostasis. A major regulator of intracellular protein distribution is the trans-Golgi network (TGN) that sorts secretory proteins into specific carriers to transport them to their final destination. The sorting of lysosomal hydrolases at the TGN by the mannose-6-phosphate receptor is well established. However, the sorting mechanism for secreted proteins remains poorly understood. The recent discovery of a Ca2+-based sorting of secretory cargo at the TGN is beginning to uncover the mechanism by which cells sort secretory cargoes from Golgi residents and cargoes destined to the other cellular compartments. This Ca2+ based sorting involves the cytoplasmic actin cytoskeleton, which through membrane-anchored Ca2+ ATPase SPCA1 and the luminal Ca2+ binding protein Cab45 sorts of a subset of secretory proteins at the TGN. The underlying mechanism regulating the interplay between these components, however, is not clear. We take an interdisciplinary and highly innovative approach to address this issue of fundamental importance. Our aims include in vitro reconstitution of these components in model membranes to identify the minimal machinery required for the sorting of secretory cargoes. Modern genome editing techniques in human cells coupled to fluorescence and super-resolution microscopy will provide new insights into the functional interconnection of the components in living cells. Altogether, our findings will reveal the mechanism by which TGN sorts and targets proteins to their respective destination, a process that is essential for cell compartmentation, tissue organization, and function.